The present invention relates to treatment and prevention of nicotine addiction. In particular, the invention relates to novel hapten-carrier conjugates which are capable of inducing the production of antibodies. Such antibodies are capable of specifically binding to nicotine. Furthermore, the present invention envisages preventing or treating nicotine addiction by administering a nicotine-carrier conjugate in a pharmaceutically-acceptable formulation. The present invention also contemplates using the antibodies raised in response to the hapten-carrier conjugate for the prevention and treatment of nicotine addiction.
Smoking of cigarettes, cigars, and pipes is a prevalent problem in the United States and worldwide. Smoking tobacco and smokeless tobacco are rich in nicotine, which is a known addictive substance. Nicotine is an alkaloid derived from the tobacco plant that is responsible for smoking""s psychoactive and addictive effects. Nicotine is formed of two rings linked together by a single bond: an aromatic six-membered ring (pyridine) and an aliphatic five-membered ring (pyrrolidine). The pyrrolidine is N-methylated and linked through its carbon-2 to the carbon-3 of pyridine. Thus, the carbon-2 is chiral, and there is virtually free rotation around the single bond linking the two rings. It has been established that the absolute configuration of carbon-2 is S. Thus, the natural configuration of nicotine is (S)-(xe2x88x92)-nicotine.
Nicotine use is widespread due to the easy availability of cigarettes, cigars, pipes and smokeless tobacco. According to the U.S. Department of Health and Human Services, cigarette smoking is the single leading cause of preventable death in the United States. See also McGinnis et al., J. Am. Med. Assoc., 270, 2207-2211 (1993). Exposure to second hand smoke also has been reported to have serious detrimental health effects, including exacerbation of asthma.
Even though the addictive nature of nicotine is well known, cigarette smoking is prevalent. Peak levels of nicotine in the blood, about 25 to 50 nanograms /ml, are achieved within 10-15 minutes of smoking a cigarette. In humans, smoking a cigarette results in arterial nicotine concentrations being 10-fold higher than venous nicotine concentrations because nicotine is rapidly delivered from the lungs to the heart (see Henningfield (1993) Drug Alcohol Depend. 33:23-29). This results in a rapid delivery of high arterial concentrations of nicotine to the brain. Once nicotine crosses the blood-brain barrier, evidence suggests that it binds to cholinergic receptors, which are normally activated by the neurotransmitter acetylcholine, which is involved in respiration, maintenance of heart rate, memory, alertness and muscle movement. When nicotine binds to these receptors, it can affect normal brain function, by triggering the release of other neurotransmitters, such as dopamine. Dopamine is found in the brain in regions involved in emotion, motivation, and feelings of pleasure. It is the release of neurotransmitters, especially dopamine, that is responsible for the tobacco user""s addiction to nicotine or other intake of nicotine.
Due to the significant adverse effects of smoking on health, smokers often try to quit. However, the addictive nature of nicotine and the availability of cigarettes add to the continued dependence on nicotine and high failure rate of those who try to quit. Withdrawal symptoms are unpleasant, and are relieved by smoking.
Many therapies for nicotine addiction have been developed, but are largely ineffective. The two most popular therapies remain the nicotine transdermal patch and nicotine incorporated into chewing gum. These therapies, termed xe2x80x9cnicotine replacement therapiesxe2x80x9d (NRT), replace the amount of nicotine which the user previously received from smoking and act to wean the user off nicotine. However, certain drawbacks are seen with this type of therapy. Particularly, there is low penetration of nicotine into the bloodstream and therefore an increased desire to smoke. Problems such as mouth irritation, jaw soreness, nausea, have been associated with use of nicotine chewing gum. Problems such as skin irritations, sleep disturbance, and nervousness have been associated with use of nicotine transdermal patches.
Therefore, an alternative methodology for treating nicotine addiction is needed. The literature recognizes this need and there have been several attempts to provide a methodology for treating nicotine addiction. One of the methods involves the administration of antibodies which have been raised in response to nicotine. However, low molecular weight substances, called haptens, are known to be unable to trigger an immune response in host animals. Nicotine is no exception, and as a small molecule it is not immunogenic. To elicit an antibody response to a hapten, it typically is covalently bound to a carrier protein, and the complex will elicit the production of antibodies, that recognize the hapten.
For example, cotinine 4xe2x80x2-carboxylic acid, when bound covalently to keyhole limpet hemocyanin (KLH) was used to generate antibodies to the nicotine metabolite cotinine. Those antibodies were used to determine the presence of cotinine in physiological fluids. See Bjerke et al. J. Immunol. Methods, 96, 239-246 (1987).
Other nicotine antibodies were prepared by Castro et al., (Eur. J. Biochem., 104, 331-340 (1980)). Castro et al. prepared nicotine haptens, conjugated to bovine serum albumin (BSA), with the carrier protein conjugated via a linker at the 6-position of nicotine. Castro et al. prepared additional nicotine conjugates of BSA which were injected into mammals to raise antibodies. In another publication, Castro et al. in Biochem. Biophys. Res. Commun. 67, 583-589 (1975) disclose two nicotine albumin conjugates: N-succinyl-6-amino-(xc2x1)-nicotine-BSA and 6-("sgr"-aminocapramido)-(xc2x1)-nicotine-BSA. In this 1975 publication, Castro et al. also used antibodies to nicotine carrier conjugate, 6-("sgr"-aminocapramido)-(xc2x1)-nicotine-BSA, to determine the levels of nicotine in blood and urine, see Res. Commun Chem. Path. Pharm. 51, 393-404 (1986).
Swain et al. (WO 98/14216) disclose nicotine carrier conjugates wherein the hapten is conjugated at the 1, 2, 4, 5, 6, or 1xe2x80x2 position of the nicotine. Hieda et al. have shown that animals immunized with 6-(carboxymethylureido)-(xc2x1)-nicotine, which was linked to keyhole limpet hemocyanin, produced antibodies specific to nicotine. J. Pharm. and Exper. Thera. 283, 1076-1081 (1997). Langone et al. prepared the hapten derivative, O-succinyl-3xe2x80x2-hydroxymethyl-nicotine, see Biochemistry, 12, 5025-5030, and used the antibodies to this hapten carrier conjugate in radioimmunoassays. See Methods in Enzymology, 84, 628-635 (1982). The conjugate produced by Langone is susceptible to hydrolysis. Additionally, Abad et al. in Anal. Chem., 65, 3227-3231 (1993) describe conjugating 3xe2x80x2-(hydroxymethyl) nicotine hemisuccinate to bovine serum albumin to produce antibodies to nicotine in order to be able to measure nicotine content in smoke condensate of cigarettes in an ELISA assay.
Therefore, the prior art does not teach a stable nicotine-carrier conjugate that preserves the chiral nature of the nicotine hapten, and that links the hapten to the carrier in a way that conserves the nature of the nicotine epitope(s). Moreover, the art does not teach or suggest methods of preventing and treating nicotine addiction by using such conjugates. Seeman in Heterocycles, 22, 165-193, (1984) discloses results of a study of the conformational analysis and chemical reactivity of nicotine.
In response to the demand for a more effective methodology for treating nicotine addiction, it is one object of the present invention to provide novel nicotine-carrier conjugates that are stable, comprise nicotine in its natural (S)-(xe2x88x92) formation, and employ a nicotine-carrier linkage that preserves the nature of the nicotine epitope(s), and the relative orientation of the two rings of the nicotine molecule. Both rings of nicotine, and their relative orientation, are believed to be essential for the recognition by antibody of nicotine in solution. Such conjugates are capable of stimulating the production of antibodies that are capable of specifically binding to nicotine. Using the inventive conjugates, the inventors have raised serum nicotine levels, and decreased brain nicotine levels, in mammals. Additionally, using the conjugates of the invention, the inventors also have prevented nicotine-induced changes in blood pressure, and locomotor effects.
In another object of the present invention is provided a method of treating nicotine addiction by administering a conjugate of the invention to a patient addicted to nicotine thereby generate anti-nicotine antibodies in that patient. Thus, when the patient smokes (or uses chewing tobacco), the nicotine from these products will be bound by the anti-nicotine antibodies in the blood, preventing the nicotine from crossing the blood-brain barrier, hence eliminating the nicotine-induced alterations in brain chemistry, which is the source of nicotine-addiction. In this regard, it is important that the nicotine-carrier conjugate elicit the production of antibodies that will recognize the native nicotine molecule. As described above, the novel nicotine-carrier conjugates of the invention preserve the chirality and the epitope(s) of naturally-occurring nicotine.
The inventors do not intend to be bound by any particular theory as to how the nicotine conjugates, and the antibodies produced in response to such conjugates, inhibit the effects of nicotine ingested by mammals. In addition to preventing nicotine from crossing the blood brain barrier, the antibodies also may prevent nicotine from binding to other receptors in the peripheral nervous system by simple steric blockage.
These objects can be achieved by providing a hapten-carrier conjugate of formula (I): 
wherein m is 1 to 2500, n is 0 to 12, y is 1 to 12, X is selected from the group consisting of NHxe2x80x94CO, COxe2x80x94NH, COxe2x80x94NHxe2x80x94NH, NHxe2x80x94NHxe2x80x94CO, NHxe2x80x94COxe2x80x94NH, COxe2x80x94NHxe2x80x94NHxe2x80x94CO, and Sxe2x80x94S; Y is selected from the group consisting of NHxe2x80x94CO, COxe2x80x94NH, COxe2x80x94NHxe2x80x94NH, NHxe2x80x94NHxe2x80x94CO, NHxe2x80x94COxe2x80x94NH, COxe2x80x94NHxe2x80x94NHxe2x80x94CO, and Sxe2x80x94S, and the xe2x80x94(CH2)nxe2x80x94Xxe2x80x94(CH2)yxe2x80x94Yxe2x80x94 moiety is bonded to the 3xe2x80x2, 4xe2x80x2 or 5xe2x80x2 position. In a preferred embodiment of the hapten-carrier conjugate, m is 11 to 17, n is 1, y is 2, X is NHxe2x80x94CO, Y is COxe2x80x94NH, the carrier protein is exoprotein A and the xe2x80x94(CH2)nxe2x80x94Xxe2x80x94(CH2)yxe2x80x94Yxe2x80x94 moiety is bonded to the 3xe2x80x2 position. In another preferred embodiment of the hapten-carrier conjugate, m is 11 to 17, n is 1, y is 2, X is NHxe2x80x94CO, Y is COxe2x80x94NH, the carrier protein is exoprotein A and the xe2x80x94(CH2)nxe2x80x94Xxe2x80x94(CH2)yxe2x80x94Yxe2x80x94 moiety is bonded to the 4xe2x80x2 position. In a further preferred embodiment of the hapten-carrier conjugate, m is 11 to 17, n is 1, y is 2, X is NHxe2x80x94CO, Y is COxe2x80x94NH, the carrier protein is exoprotein A and the xe2x80x94(CH2)nxe2x80x94Xxe2x80x94(CH2)yxe2x80x94Yxe2x80x94 moiety is bonded to the 5xe2x80x2 position. In an additionally preferred embodiment, m is selected from the group consisting of 1 to 20 and 1 to 200.
The above objects also be achieved by providing a hapten-carrier conjugate of formula (III): 
wherein n is 0 to 12, j is 1 to 1000, k is 1 to 20, and E is an amino acid-containing matrix. In a preferred embodiment, the matrix is poly-L-glutamic acid.
The objects can also be achieved by providing an antibody which is produced in response to the hapten-carrier conjugate of Formula (I). In an additional embodiment, the antibody is a functional fragment. In a preferred embodiment, the antibody is a monoclonal antibody. In an additional embodiment of the invention, the antibody is polyclonal.
The objects can also be achieved by providing an antibody which is produced in response to the hapten-carrier conjugate of Formula (III). In an additional embodiment, the antibody is a functional fragment. In a preferred embodiment, the antibody is a monoclonal antibody. In an additional embodiment of the invention, the antibody is polyclonal.
The objects can be achieved by providing a method of treating or preventing nicotine addiction in a patient in need of such treatment comprising administering a therapeutically effective amount of the hapten-carrier conjugate of Formula (I) or (III). Alternatively, the objects can be achieved by providing a method treating or preventing nicotine addiction in a patient in need of such treatment comprising administering a therapeutically effective amount of antibody raised in response to the hapten-carrier conjugates of Formula (I) or (III).
Additionally, the objects can be achieved by providing a vaccine composition which comprises the hapten carrier conjugate of Formula (I) or Formula (III). In addition the vaccine can further comprise an additional therapeutic compound for treating nicotine addiction.
The objects also can be achieved by providing a process for producing an antibody, comprising immunizing a host mammal with a hapten-carrier conjugate of Formula (I) or (III). In a preferred embodiment, the antibody produced is a monoclonal antibody. In an additional embodiment the antibody is polyclonal.
Additional objects can be achieved by providing a kit for determining the presence of nicotine in a sample, comprising an antibody of raised in response to the hapten-carrier conjugate of Formula (I) or Formula (III).
These objects and others apparent to those skilled in the art have been achieved by the invention described below in the detailed description and appended claims.